中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2015年
4期
843-846
,共4页
蔡峰%韦继南%谢鑫荟%王锋%朱磊%王小虎%时睿%王琨%吴小涛
蔡峰%韋繼南%謝鑫薈%王鋒%硃磊%王小虎%時睿%王琨%吳小濤
채봉%위계남%사흠회%왕봉%주뢰%왕소호%시예%왕곤%오소도
基质细胞衍生因子-1%趋化因子受体4%椎间盘退变%迁移
基質細胞衍生因子-1%趨化因子受體4%椎間盤退變%遷移
기질세포연생인자-1%추화인자수체4%추간반퇴변%천이
Stromal cell-derived factor-1%Chemokine receptor 4%Intervertebral disc degeneration%Migration
目的 探讨骨髓间充质干细胞(BMSCs)在椎间盘中的迁移,以及基质细胞衍生因子-1/趋化因子受体4 (SDF-1/CXCR4)信号轴对BMSCs迁移的调控.方法 分离培养兔BMSCs和髓核细胞(NPCs)并鉴定.Transwell迁移实验,A组:未处理的BMSCs;B组:过表达CXCR4的BMSCs,C组:CXCR4特异性阻断剂普乐沙福(AMD3100,5 mg/L)处理30 min后的BMSCs;其中A、B、C3组将BMSCs与下室NPCs共培养,D组BMSCs单独培养.髓核抽吸法造模,2周后移植绿色荧光蛋白(GFP)标记的BMSCs,移植后12周取椎间盘组织,随机分成甲、乙两组,甲组用免疫荧光法标记SDF-1和GFP,乙组标记CXCR4和GFP.分别计算髓核(NP)、交界区和纤维环(AF)中SDF-1阳性细胞占椎间盘细胞的比例以及观察GFP与CXCR4共表达细胞的分布规律.结果 P3代BMSCs表面分子CD29、CD90和CD34的表达率分别为(96.8±2.2)％、(96.1±1.9)％和(1.5±0.6)％,并具有三系分化能力.P1代NPCs表达Ⅱ型胶原(COL2)和聚集蛋白聚糖(ACAN).迁移实验中A、B、C、D4组膜下细胞数分别为(70.30±3.25)、(86.70±1.71)、(51.30±1.63)、(29.70±2.08)个.迁移能力依次为:B＞A＞C＞D组.免疫荧光结果显示BMSCs主要分布在NP中央区,由中央区到交界区,BMSCs数量逐渐减少,NP中央区和交界区BMSCs的CXCR4表达率分别为(41.19±7.20)％和(27.68 ±4.30)％.NP、交界区和AF中椎间盘细胞SDF-1表达率分别为(84.02±3.20)％、(71.48±5.90)％和(49.31±4.60)％.结论 BMSCs移植到椎间盘后从移植部位向退变NPCs迁移.SDF-1/CXCR4信号轴是促使BMSCs向退变NPCs迁移的重要机制之一.
目的 探討骨髓間充質榦細胞(BMSCs)在椎間盤中的遷移,以及基質細胞衍生因子-1/趨化因子受體4 (SDF-1/CXCR4)信號軸對BMSCs遷移的調控.方法 分離培養兔BMSCs和髓覈細胞(NPCs)併鑒定.Transwell遷移實驗,A組:未處理的BMSCs;B組:過錶達CXCR4的BMSCs,C組:CXCR4特異性阻斷劑普樂沙福(AMD3100,5 mg/L)處理30 min後的BMSCs;其中A、B、C3組將BMSCs與下室NPCs共培養,D組BMSCs單獨培養.髓覈抽吸法造模,2週後移植綠色熒光蛋白(GFP)標記的BMSCs,移植後12週取椎間盤組織,隨機分成甲、乙兩組,甲組用免疫熒光法標記SDF-1和GFP,乙組標記CXCR4和GFP.分彆計算髓覈(NP)、交界區和纖維環(AF)中SDF-1暘性細胞佔椎間盤細胞的比例以及觀察GFP與CXCR4共錶達細胞的分佈規律.結果 P3代BMSCs錶麵分子CD29、CD90和CD34的錶達率分彆為(96.8±2.2)％、(96.1±1.9)％和(1.5±0.6)％,併具有三繫分化能力.P1代NPCs錶達Ⅱ型膠原(COL2)和聚集蛋白聚糖(ACAN).遷移實驗中A、B、C、D4組膜下細胞數分彆為(70.30±3.25)、(86.70±1.71)、(51.30±1.63)、(29.70±2.08)箇.遷移能力依次為:B＞A＞C＞D組.免疫熒光結果顯示BMSCs主要分佈在NP中央區,由中央區到交界區,BMSCs數量逐漸減少,NP中央區和交界區BMSCs的CXCR4錶達率分彆為(41.19±7.20)％和(27.68 ±4.30)％.NP、交界區和AF中椎間盤細胞SDF-1錶達率分彆為(84.02±3.20)％、(71.48±5.90)％和(49.31±4.60)％.結論 BMSCs移植到椎間盤後從移植部位嚮退變NPCs遷移.SDF-1/CXCR4信號軸是促使BMSCs嚮退變NPCs遷移的重要機製之一.
목적 탐토골수간충질간세포(BMSCs)재추간반중적천이,이급기질세포연생인자-1/추화인자수체4 (SDF-1/CXCR4)신호축대BMSCs천이적조공.방법 분리배양토BMSCs화수핵세포(NPCs)병감정.Transwell천이실험,A조:미처리적BMSCs;B조:과표체CXCR4적BMSCs,C조:CXCR4특이성조단제보악사복(AMD3100,5 mg/L)처리30 min후적BMSCs;기중A、B、C3조장BMSCs여하실NPCs공배양,D조BMSCs단독배양.수핵추흡법조모,2주후이식록색형광단백(GFP)표기적BMSCs,이식후12주취추간반조직,수궤분성갑、을량조,갑조용면역형광법표기SDF-1화GFP,을조표기CXCR4화GFP.분별계산수핵(NP)、교계구화섬유배(AF)중SDF-1양성세포점추간반세포적비례이급관찰GFP여CXCR4공표체세포적분포규률.결과 P3대BMSCs표면분자CD29、CD90화CD34적표체솔분별위(96.8±2.2)％、(96.1±1.9)％화(1.5±0.6)％,병구유삼계분화능력.P1대NPCs표체Ⅱ형효원(COL2)화취집단백취당(ACAN).천이실험중A、B、C、D4조막하세포수분별위(70.30±3.25)、(86.70±1.71)、(51.30±1.63)、(29.70±2.08)개.천이능력의차위:B＞A＞C＞D조.면역형광결과현시BMSCs주요분포재NP중앙구,유중앙구도교계구,BMSCs수량축점감소,NP중앙구화교계구BMSCs적CXCR4표체솔분별위(41.19±7.20)％화(27.68 ±4.30)％.NP、교계구화AF중추간반세포SDF-1표체솔분별위(84.02±3.20)％、(71.48±5.90)％화(49.31±4.60)％.결론 BMSCs이식도추간반후종이식부위향퇴변NPCs천이.SDF-1/CXCR4신호축시촉사BMSCs향퇴변NPCs천이적중요궤제지일.
Objective To investigate the migration of bone marrow mesenchymal stem cells (BMSCs) in degenerated intervertebral discs,and the role of stromal cell-derived factor-1/CXC chemokine receptor4 (SDF-1/CXCR4) axis in this process.Methods BMSCs and nucleus pulposus cells (NPCs) were isolated and expanded.BMSCs were authenticated by flow cytometry and multilineage differentiation,and NPCs were authenticated by toluidine blue and immunocytochemistry staining.The transwell was used to evaluate migration ability,group A:normal BMSCs + NPCs ; group B:CXCR4 over-expression BMSCs + NPCs; group C:BMSCs with blockage of CXCR4 by Plerixafor (AMD3100) + NPCs ; group D:normal BMSCs alone.BMSCs marked by green fluorescent protein (GFP) were transplanted into discs 2 weeks after disc degeneration induction.Discs were collected 12 weeks after cell transplantation,and randomly divided into 2 groups.Group one:detect SDF-1 and GFP by immunofluorescence; group two:detect CXCR4 and GFP.Results The mean ± SD percentages of CD29-positive,CD90-positive and CD34-positive cells in total BMSCs were (96.8 ± 2.2) ％,(96.1 ± 1.9) ％ and (1.5 ± 0.6) ％ respectively.NPCs expressed COL2 and ACAN.The migration ability in transwll is group B ＞ A ＞ C ＞ D.The result of immunofluorescence showed that BMSCs were distributed in discs.The mean ± SD percentages of CXCR4-positive cells in total BMSCs in the center of nucleus pulposus and transition area were (41.19 ± 7.20) ％ and (27.68 ± 4.30) ％ respectively.The mean ± SD percentages of SDF-1-positive cells in total disc cells in the center of nucleus pulposus,transition area and annulus fibrosus were (84.02 ± 3.20)％,(71.48±5.90)％ and (49.31 ±4.60)％ respectively.Conclusion BMSCs can migrate to degenerated NPCs in intervertebral discs.The SDF-1/CXCR4 axis improves the migration of BMSCs transplanted into degenerated intervertebral discs.
